Compositions and methods to detect Legionella pneumophila nucleic acid

ABSTRACT

Compositions are disclosed as nucleic acid sequences that may be used as amplification oligomers, including primers, capture probes for sample preparation, and detection probes specific for  Legionella pneumophila  16S or 23S rRNA sequences or DNA encoding 16S or 23S rRNA. Methods are disclosed for detecting the presence of  L. pneumophila  in samples by using the disclosed compositions in methods that include in vitro nucleic acid amplification of a 16S rRNA sequence or DNA encoding the 16S rRNA sequence, or of a 23S rRNA sequence or DNA encoding the 23S rRNA sequence to produce a detectable amplification product.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/075,944,filed Nov. 8, 2013 and now allowed, which is a continuation ofapplication Ser. No. 12/624,233, filed Nov. 23, 2009, now U.S. Pat. No.8,609,829, which is a continuation-in-part of application Ser. No.11/582,770, filed Oct. 17, 2006 and now abandoned, which claims thebenefit under 35 U.S.C. 119(e) of provisional applications nos.60/727,883, filed Oct. 17, 2005, and 60/735,709, filed Nov. 9, 2005, allof which are incorporated by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Nov. 8, 2017, isnamed “GP190-06-CN2_ST25.txt” and is 24 KB in size.

FIELD

This invention relates to detection of the presence of bacteria in asample by using molecular biological methods, and specifically relatesto detection of Legionella pneumophila in a sample by amplifying L.pneumophila nucleic acid sequences and detecting the amplified nucleicacid sequences.

BACKGROUND

Legionellae, which consists of the one genus Legionella, are fastidiousgram-negative bacteria found in moist environments as intracellularparasites of freshwater protozoa (Fields, et al., 2002, Clin. Microbiol.Rev. 15(3): 506-526). Legionellae can multiply in mammalian cells andcause respiratory disease in humans when a susceptible host inhales oraspirates water or an aerosol containing the bacteria. Although at least48 species of Legionella are known, L. pneumophila is responsible formost reported cases of legionellosis that result in a severe multisystemdisease involving pneumonia, and most other legionellosis cases arecaused by L. bozemanii, L. dumoffli, L. longbeachae, and L. micdadei.

Legionellae may be detected from a number of specimen types and by usinga variety of methods. Culture of bacteria from bronchoscopy,bronchoalveolar lavage (BAL), or lung biopsy specimens in a specializedBuffered Charcoal Yeast Extract medium (BCYE) is sensitive and accuratebut requires up to two weeks of incubation for maximal recovery followedby identification of the bacteria by using a combination of colonymorphology, gram staining, and serologic testing, e.g., immunoassays.Although direct detection of Legionella in uncultured clinical specimensis possible by immunofluorescent or radioimmunoassay methods, thesetests are often less sensitive. Legionellosis may be diagnosed byindirect detection of a soluble polysaccharide antigen of L. pneumophilaserogroup 1 in urine, but these assays have limited diagnostic utilitybecause of the time delay needed for seroconversion and cannot detect byused for environmental testing. Molecular diagnostic tests have beendeveloped that use DNA probes or a combination of nucleic acidamplification and DNA probes to detect genetic sequences of Legionellae,including the mip gene of L. pneumophila. Such methods detect thepresence of nucleic acids from Legionellae in a variety of specimens andwith varying degrees of specificity and sensitivity. Many such tests,however, are labor intensive, require at least a day to perform, and aresubject to contamination that results in false positive results.

Because Legionellae can survive and persist for a long time in aquaticand moist environments, such as reservoirs and cooling tower water, theycan cause community acquired or nosocomial infections. Hence, there is aneed for a rapid, sensitive and accurate method to detect Legionellae,particularly L. pneumophila, in environmental samples so that aninfectious source can be accurately detected and eliminated to preventinfections. There is also a need for methods that allow rapid andaccurate detection of L. pneumophila infections in humans so thatinfected people may be treated promptly to limit morbidity, mortality,and spread of infection.

SUMMARY

Disclosed are methods of detecting Legionella pneumophila in a sample,including environmental samples or biological specimens derived frominfected humans, by amplifying and detecting target sequences containedin L. pneumophila 16S rRNA or 23S rRNA, of DNA encoding them. By usingspecific primers and probes disclosed herein, the methods amplify targetsequences in 16S and/or 23S rRNA sequences of L. pneumophila and detectthe amplified products. Some embodiments monitor the development ofspecific amplification products during the amplification step whereasother embodiments detect the amplification products following theamplification step. Some method embodiments include detection of aninternal control or calibrator, e.g., a non-Legionella sequence.

A method is disclosed for detecting L. pneumophila in a sample thatincludes the steps of: providing a sample that contains a L. pneumophilatarget nucleic acid that is a 16S rRNA sequence or DNA encoding the 16SrRNA sequence, mixing the sample with at least one first amplificationoligonucleotide selected from the group consisting of SEQ ID NOS. 30,31, 32, 33, 34, 35, 36, 37, 40, 41, 42, 43, 44, 45, 53, 54, 60 and 61,combined with at least one second amplification oligonucleotide selectedfrom the group consisting of SEQ ID NOS. 28, 29, 38, 39, 46, 47, 48, 49,50, 51, 52, 55, 56, 57, 58, and 59, providing an enzyme with nucleicacid polymerase activity and nucleic acid precursors to make anamplification mixture that includes the first and second amplificationoligonucleotides and the L. pneumophila target nucleic acid, elongatingin vitro a 3′ end of at least one of the amplification oligonucleotideshybridized to the L. pneumophila target nucleic acid by using the enzymewith nucleic acid polymerase activity and the L. pneumophila targetnucleic acid as a template to produce an amplified product, anddetecting the amplified product to indicate the presence Legionellapneumophila in the sample. In some embodiments, the detecting stephybridizes the amplified product specifically to a detection probeoligomer consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 62, 63, 64, or65. Some embodiments also include a sample processing step that capturesthe L. pneumophila target nucleic acid from the sample before thehybridizing step, preferably by using a capture probe oligomer thatcontains a target specific sequence consisting of SEQ ID NO:66, SEQ IDNO:67 or SEQ ID NO:68, which may be covalently attached to a 3′ tailsequence. In some embodiments, the mixing step uses a combination of thefirst and second amplification oligonucleotides selected from the groupconsisting of: SEQ ID NO:29 with SEQ ID NO:31, SEQ ID NO:28 with SEQ IDNO:31, SEQ ID NO:29 with SEQ ID NO:33, SEQ ID NO:28 with SEQ ID NO:33,SEQ ID NO: 41 with SEQ ID NO:46, SEQ ID NO:41 with SEQ ID NO:55, SEQ IDNO:54 with SEQ ID NO:46, SEQ ID NO:54 with SEQ ID NO:55, SEQ ID NO:51with SEQ ID NO:43, SEQ ID NO:52 with SEQ ID NO:43, SEQ ID NO:51 with SEQID NO:45, SEQ ID NO:52 with SEQ ID NO:45, SEQ ID NO:60 with SEQ ID NO:58and SEQ ID NO:56, SEQ ID NO:60 with SEQ ID NO:59 and SEQ ID NO:56, SEQID NO:60 with SEQ ID NO:58 and SEQ ID NO:57, SEQ ID NO:60 with SEQ IDNO:59 and SEQ ID NO:57, SEQ ID NO:61 with SEQ ID NO:58 and SEQ ID NO:56,SEQ ID NO:61 with SEQ ID NO:59 and SEQ ID NO:56, SEQ ID NO:61 with SEQID NO:58 and SEQ ID NO:57, and SEQ ID NO:61 with SEQ ID NO:59 and SEQ IDNO:57. In some preferred embodiments, the mixing step uses a combinationof the first and second amplification oligonucleotides selected from thegroup consisting of: SEQ ID NO:29 with SEQ ID NO:31, SEQ ID NO:28 withSEQ ID NO:31, SEQ ID NO: 41 with SEQ ID NO:46, SEQ ID NO:41 with SEQ IDNO:55, SEQ ID NO:54 with SEQ ID NO:46, SEQ ID NO:54 with SEQ ID NO:55,SEQ ID NO:52 with SEQ ID NO:43, and SEQ ID NO:52 with SEQ ID NO:45.

A composition is disclosed for detecting Legionella pneumophila 16S rRNAsequence or DNA encoding the 16S rRNA sequence by using in vitroamplification, that includes at least one first amplificationoligonucleotide selected from the group consisting of SEQ ID NOS. 30,31, 32, 33, 34, 35, 36, 37, 40, 41, 42, 43, 44, 45, 53, 54, 60 and 61,combined with at least one second amplification oligonucleotide selectedfrom the group consisting of SEQ ID NOS. 28, 29, 38, 39, 46, 47, 48, 49,50, 51, 52, 55, 56, 57, 58, and 59. The composition may also include atleast one capture probe oligomer that contains a target specificsequence consisting of SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:68, whichis optionally linked with a 3′ tail sequence. The composition may alsoinclude at least one detection probe oligomer selected from the groupconsisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 62, 63, 64, and 65.Preferred embodiments include at least one detection probe oligomerselected from the group consisting of SEQ ID NOS. 5, 13, 15, and 21.Preferred embodiments of such compositions are provided in the form of akit, which may optionally include other reagents used in nucleic acidamplification and/or detection.

A method is disclosed for detecting Legionella pneumophila in a samplethat includes the steps of providing a sample that contains a L.pneumophila target nucleic acid that is a 23S rRNA sequence or DNAencoding the 23S rRNA sequence, mixing the sample with at least onefirst amplification oligonucleotide selected from the group consistingof SEQ ID NOS. 69, 70, 71, 74, 75, 76, 77, 78, 79, 80, 81, 82, and 83,combined with at least one second amplification oligonucleotide selectedfrom the group consisting of SEQ ID NOS. 84, 85, 86 and 87, providing anenzyme with nucleic acid polymerase activity and nucleic acid precursorsto make an amplification mixture that includes the first and secondamplification oligonucleotides and the L. pneumophila target nucleicacid, elongating in vitro a 3′ end of at least one of the amplificationoligonucleotides hybridized to the L. pneumophila target nucleic acid byusing the enzyme with nucleic acid polymerase activity and the L.pneumophila target nucleic acid as a template to produce an amplifiedproduct, and detecting the amplified product to indicate the presenceLegionella pneumophila in the sample. In some embodiments, the detectingstep hybridizes the amplified product specifically to a detection probeoligomer selected from the group consisting of SEQ ID NOS. 72, 88 and89. Other embodiments may also include a sample processing step thatcaptures the L. pneumophila target nucleic acid from the sample beforethe hybridizing step, preferably by using a capture probe oligomer thatcontains a target specific sequence consisting of SEQ ID NO:73, whichmay be covalently attached to a 3′ tail sequence. In some embodiments,the mixing step uses a combination of the first and second amplificationoligonucleotides selected from the group consisting of: SEQ ID NO:69 orSEQ ID NO:70 with SEQ ID NO:84, any one of SEQ ID NOS. 71 to 77 with SEQID NO:84, SEQ ID NO:78 or SEQ ID NO:79 with SEQ ID NO:84, any one of SEQID NOS. 80 to 83 with SEQ ID NO:84, SEQ ID NO:69 or SEQ ID NO:70 withSEQ ID NO:85, any one of SEQ ID NOS. 71 to 77 with SEQ ID NO:85, SEQ IDNO:78 or SEQ ID NO:79 with SEQ ID NO:85, any one of SEQ ID NOS. 80 to 83with SEQ ID NO:85, SEQ ID NO:69 or SEQ ID NO:70 with SEQ ID NO:86, anyone of SEQ ID NOS. 71 to 77 with SEQ ID NO:86, SEQ ID NO:78 or SEQ IDNO:79 with SEQ ID NO:86, any one of SEQ ID NOS. 80 to 83 with SEQ IDNO:86, SEQ ID NO:69 or SEQ ID NO:70 with SEQ ID NO:87, any one of SEQ IDNOS. 71 to 77 with SEQ ID NO:87, SEQ ID NO:78 or SEQ ID NO:79 with SEQID NO:87, and any one of SEQ ID NOS. 80 to 83 with SEQ ID NO:87.Preferred embodiments include those in which the mixing step uses acombination of the first and second amplification oligonucleotidesselected from the group consisting of: SEQ ID NO:79 with SEQ ID NO:85and SEQ ID NO:87; SEQ ID NO:75 with SEQ ID NO:84 and SEQ ID NO:87; andSEQ ID NO:75 with SEQ ID NO:85 and SEQ ID NO:87.

A composition is disclosed for detecting a Legionella pneumophila 23SrRNA sequence or DNA encoding the 23S rRNA sequence by using in vitroamplification, that includes at least one first amplificationoligonucleotide selected from the group consisting of SEQ ID NOS. 69,70, 71, 74, 75, 76, 77, 78, 79, 80, 81, 82, and 83, combined with atleast one second amplification oligonucleotide selected from the groupconsisting of SEQ ID NOS. 84, 85, 86 and 87. In preferred embodiments,the composition is a combination of first and second amplificationoligonucleotides selected from the group consisting of: SEQ ID NO:79with SEQ ID NO:85 and SEQ ID NO:87; SEQ ID NO:75 with SEQ ID NO:84 andSEQ ID NO:87; and SEQ ID NO:75 with SEQ ID NO:85 and SEQ ID NO:87. Someembodiments also include at least one capture probe oligomer thatcontains a target specific sequence consisting of SEQ ID NO:73, whichmay be covalently attached to a 3′ tail sequence. Some embodiments ofthe composition also include at least one detection probe oligomerselected from the group consisting of SEQ ID NOS. 72, 88 and 89.Preferred embodiments of such compositions are provided in the form of akit, which may optionally include other reagents used in nucleic acidamplification and/or detection.

DETAILED DESCRIPTION

Methods are disclosed for sensitively and specifically detecting thepresence of L. pneumophila in an environmental or biological sample bydetecting L. pneumophila nucleic acids. The methods include performing anucleic acid amplification of 16S or 23S rRNA sequences and detectingthe amplified product, typically by using a nucleic acid probe thatspecifically hybridizes to the amplified product to provide a signalthat indicates the presence of L. pneumophila in the sample. Theamplification step includes contacting the sample with a one or moreamplification oligomers specific for a target sequence in 16S or 23SrRNA to produce an amplified product if L. pneumophila rRNA in presentin the sample. Amplification synthesizes additional copies of the targetsequence or its complement by using at least one nucleic acid polymeraseto extend the sequence from an amplification oligomer (a primer) using aL. pneumophila template strand. Preferred embodiments for detecting theamplified product use a hybridizing step that includes contacting theamplified product with at least one probe specific for an amplifiedsequence, e.g., a sequence contained in the target sequence that isflanked by a pair of amplification oligomers. The detecting step may beperformed after the amplification reaction is completed, or may beperformed simultaneous with the amplification reaction (sometimesreferred to as “real time”). In preferred embodiments, the detectionstep detects the amplified product that uses a probe that is detected ina homogeneous reaction, i.e., detection of the hybridized probe does notrequire removal of unhybridized probe from the mixture (e.g., U.S. Pat.Nos. 5,639,604 and 5,283,174, Arnold Jr. et al.). In preferredembodiments that detect the amplified product near or at the end of theamplification step, a linear probe hybridizes to the amplified productto provide a signal that indicates hybridization of the probe to theamplified sequence. In preferred embodiments that use real-timedetection, the probe is preferably a hairpin structure probe thatincludes a reporter moiety that provides the detected signal when theprobe binds to the amplified product. For example, a hairpin probe mayinclude a reporter moiety or label, such as a fluorophore (“F”),attached to one end of the probe and an interacting compound, such asquencher (“Q”), attached to the other end the hairpin structure toinhibit signal production when the hairpin structure is in the “closed”conformation and not hybridized to the amplified product, whereas adetectable signal results when the probe is hybridized to acomplementary sequence in the amplified product, thus converting theprobe to a “open” conformation. Examples of hairpin structure probeinclude a molecular beacon, molecular torch, or hybridization switchprobe and other forms (e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728,Lizardi et al., U.S. Pat. Nos. 5,925,517 and 6,150,097, Tyagi et al.,U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, Becker etal., U.S. Pat. No. 8,034,554, Becker et al., and US Pub. No.2006-0194240 A1, Arnold Jr. et al.).

To aid in understanding this disclosure, some terms used herein aredescribed below. Unless otherwise described, scientific and technicalterms used herein have the same meaning as commonly understood by thoseskilled in the relevant art based on technical literature, e.g., inDictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton etal., 1994, John Wiley & Sons, New York, N.Y.), The Harper CoffinsDictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York,N.Y.), or Dorland's Illustrated Medical Dictionary, 30^(th) ed. (2003,W.B. Saunders, Elsevier Inc., Philadelphia, Pa.). Unless otherwisedescribed, techniques employed or contemplated herein are standardmethods well known in the art of molecular biology.

“Sample” includes any specimen that may contain Legionella bacteria orcomponents thereof, such as nucleic acids or nucleic acid fragments.Samples may be obtained from environmental sources, e.g., water, soil,slurries, debris, biofilms from containers of aqueous fluids, airborneparticles or aerosols, and the like, which may include processedsamples, such as those obtained from passing an environmental sampleover or through a filters, by centrifugation, or by adherence to amedium, matrix, or support. “Biological samples” include any tissue ormaterial derived from a living or dead mammal, including humans, whichmay contain Legionellae or target nucleic acid derived therefrom, e.g.,respiratory tissue or exudates such as bronchoscopy, bronchoalveolarlavage (BAL) or lung biopsy, sputum, peripheral blood, plasma, serum,lymph node, gastrointestinal tissue, urine, exudates, or other bodyfluids. A sample may be treated to physically or mechanically disruptaggregates or cells to release intracellular components, includingnucleic acids, into a solution which may contain other components, suchas enzymes, buffers, salts, detergents and the like.

“Nucleic acid” refers to a multimeric compound comprising nucleosides ornucleoside analogs which have nitrogenous heterocyclic bases, or baseanalogs, which are linked by phosphodiester bonds or other linkages toform a polynucleotide. Nucleic acids include RNA, DNA, or chimericDNA-RNA polymers, and analogs thereof. A nucleic acid “backbone” may bemade up of a variety of linkages, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid (PNA) bonds (PCT No.WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, orcombinations thereof. Sugar moieties of the nucleic acid may be eitherribose or deoxyribose, or similar compounds having known substitutions,e.g., 2′ methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine; The Biochemistry of the Nucleic Acids 5-36,Adams et al., ed., 11^(th) ed., 1992), derivatives of purine orpyrimidine bases, e.g., N⁴-methyl deoxyguanosine, deaza- or aza-purines,deaza- or aza-pyrimidines, pyrimidine bases having substituent groups atthe 5 or 6 position, purine bases having an altered or replacementsubstituent at the 2, 6 and/or 8 position, such as2-amino-6-methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO⁴-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or3-substituted pyrazolo[3,4-d]pyrimidine (U.S. Pat. Nos. 5,378,825,6,949,367 and PCT No. WO 93/13121). Nucleic acids may include “abasic”positions in which the backbone does not include a nitrogenous base forone or more residues (U.S. Pat. No. 5,585,481). A nucleic acid maycomprise only conventional sugars, bases, and linkages as found in RNAand DNA, or may include conventional components and substitutions (e.g.,conventional bases linked by a 2′ methoxy backbone, or a nucleic acidincluding a mixture of conventional bases and one or more base analogs).Nucleic acids also include “locked nucleic acids” (LNA), an analoguecontaining one or more LNA nucleotide monomers with a bicyclic furanoseunit locked in an RNA mimicking sugar conformation, which enhanceshybridization affinity toward complementary sequences in single-strandedRNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA)Nester et al., 2004, Biochemistry 43(42):13233-41). Methods forsynthesizing nucleic acids in vitro are well known in the art.

The interchangeable terms “oligomer” and “oligonucleotide” refer to anucleic acid having generally less than 1,000 nucleotides (nt),including polymers in a range having a lower limit of about 2 nt to 5 ntand an upper limit of about 500 nt to 900 nt. Preferred oligomers are ina size range having a lower limit of about 5 nt to 15 nt and an upperlimit of about 50 nt to 600 nt, and particularly preferred embodimentsare in a range having a lower limit of about 10 nt to 20 nt and an upperlimit of about 22 nt to 100 nt. Preferred oligomers are synthesized byusing any well known enzymatic or chemical method and purified bystandard methods, e.g., chromatography.

An “amplification oligomer” is an oligonucleotide that hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. An example of an amplification oligomer isa “primer” that hybridizes to a template nucleic acid and contains a 3′hydroxyl end that is extended by a polymerase in an amplificationprocess. Another example is an oligonucleotide that participates in orfacilitates amplification but is not extended by a polymerase, e.g.,because it has a 3′ blocked end. Preferred size ranges for amplificationoligomers include those that are about 10 to about 60 nt long andcontain at least about 10 contiguous bases, and more preferably at least12 contiguous bases that are complementary to a region of the targetnucleic acid sequence (or its complementary sequence). The contiguousbases are preferably at least 80%, more preferably at least 90%, andmost preferably about 100% complementary to the target sequence to whichthe amplification oligomer binds. An amplification oligomer mayoptionally include modified nucleotides or analogs, or optionally anadditional sequence that participate in an amplification reaction butare not complementary to or contained in or complementary to the targetor template sequence. For example, a “promoter primer” is anoligonucleotide that includes a 5′ promoter sequence that isnon-complementary to the target nucleic acid but is adjacent or near tothe target complementary sequence of the primer. Those skilled in theart will understand that an amplification oligomer that functions as aprimer may be modified to include a 5′ promoter sequence, and thusfunction as a promoter-primer, and a promoter-primer can function as aprimer independent of its promoter sequence, i.e., the oligonucleotidemay be modified by removal of, or synthesis without, its promotersequence. An amplification oligomer referred to as a “promoter provider”includes a promoter sequence that serves as a template forpolymerization but the oligonucleotide is not extended from its 3′ endwhich is blocked and, therefore, not available for extension bypolymerase activity.

“Amplification” refers to any known in vitro procedure for obtainingmultiple copies of a target nucleic acid sequence or fragments thereof,or its complementary sequence. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., by using anamplification oligonucleotide that hybridizes to and initiatespolymerization from an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, the polymerase chain reaction (PCR), ligase chainreaction (LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600,Kramer et al.). PCR amplification uses a DNA polymerase, pairs ofprimers, and thermal cycling to synthesize multiple copies of twocomplementary strands of a dsDNA or from a cDNA (e.g., U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159, Mullis et al.). LCR amplificationuses four or more different oligonucleotides to amplify a target and itscomplementary strand by using multiple cycles of hybridization,ligation, and denaturation (e.g., U.S. Pat. No. 5,427,930, Birkenmeyeret al., U.S. Pat. No. 5,516,663, Backman et al.). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, whereby amplification occurs in a seriesof primer extension and strand displacement steps (e.g., U.S. Pat. No.5,422,252, Walker et al., U.S. Pat. No. 5,547,861, Nadeau et al., U.S.Pat. No. 5,648,211, Fraiser et al.).

“Transcription-associated amplification” or “transcription-mediatedamplification” (TMA) refer to any type of nucleic acid amplificationthat uses an RNA polymerase to produce multiple RNA transcripts from anucleic acid template. These methods generally use an RNA polymerase, aDNA polymerase, nucleic acid substrates (dNTPs and rNTPs), and atemplate complementary oligonucleotide that includes a promotersequence, and optionally may include one or more other oligonucleotides.Variations of transcription-associated amplification are well known inthe art (e.g., disclosed in detail in U.S. Pat. Nos. 5,399,491 and5,554,516, Kacian et al.; U.S. Pat. No. 5,437,990, Burg et al.; PCT Nos.WO 88/01302 and WO 88/10315, Gingeras et al.; U.S. Pat. No. 5,130,238,Malek et al.; U.S. Pat. Nos. 4,868,105 and 5,124,246, Urdea et al.; PCTNo. WO 95/03430, Ryder et al.; and U.S. Pat. No. 7,374,885, Becker etal.). TMA methods of Kacian et al. and a one-primertranscription-associated method (U.S. Pat. No. 7,374,885, Becker et al.)are preferred embodiments of transcription associated amplificationmethods for use in detection of Legionella target sequences as describedherein. Although preferred embodiments are illustrated by suchamplification reactions, a person of ordinary skill in the art willappreciated that amplification oligomers disclosed herein may be readilyused in other amplification methods that extend a sequence fromprimer(s) by using a polymerase.

“Probe” refers to a nucleic acid oligomer that hybridizes specificallyto a target sequence in a nucleic acid, preferably in an amplifiednucleic acid, under conditions that allow hybridization to permitdetection of the target sequence or amplified nucleic acid. Detectionmay either be direct (i.e., probe hybridized directly to its targetsequence) or indirect (i.e., probe linked to its target via anintermediate molecular structure). A probe's “target sequence” generallyrefers to a subsequence within a larger sequence (e.g., a subset of anamplified sequence) that hybridizes specifically to at least a portionof a probe by standard base pairing. A probe may include target-specificsequence and other sequences that contribute to the probe'sthree-dimensional conformation (e.g., described in U.S. Pat. Nos.5,118,801 and 5,312,728, Lizardi et al.; U.S. Pat. Nos. 6,849,412,6,835,542, 6,534,274, 6,361,945, and 8,034,554).

By “sufficiently complementary” is meant a contiguous sequence that iscapable of hybridizing to another sequence by hydrogen bonding between aseries of complementary bases, which may be complementary at eachposition in the sequence by standard base pairing (e.g., G:C, A:T or A:Upairing) or may contain one or more positions, including abasic ones,which are not complementary bases by standard hydrogen bonding.Contiguous bases are at least 80%, preferably at least 90%, and morepreferably about 100% complementary to a sequence to which an oligomeris intended to specifically hybridize. Sequences that are “sufficientlycomplementary” allow stable hybridization of a nucleic acid oligomer toits target sequence under the selected hybridization conditions, even ifthe sequences are not completely complementary. Appropriatehybridization conditions are well known in the art, can be predictedreadily based on base sequence composition, or can be determined byusing routine testing (e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

“Sample preparation” refers to any steps or methods that prepare asample for subsequent amplification and detection of Legionella nucleicacids present in the sample. Sample preparation may include any knownmethod of concentrating components from a larger sample volume or from asubstantially aqueous mixture, e.g., by filtration or trapping ofairborne particles from an air sample or microbes from a water sample.Sample preparation may include lysis of cellular components and removalof debris, e.g., by filtration or centrifugation, and may include use ofnucleic acid oligomers to selectively capture the target nucleic acidfrom other sample components.

A “capture probe” or “capture oligomer” refers to at least one nucleicacid oligomer that joins a target sequence and an immobilized oligomerby using base pair hybridization to selectively capture the targetsequence. A preferred capture probe embodiment includes two bindingregions: a target sequence-binding region and an immobilizedprobe-binding region, usually on the same oligomer, although the tworegions may be present on different oligomers joined by one or morelinkers. For example, a first oligomer may include the immobilizedprobe-binding region and a second oligomer may include the targetsequence-binding region, and the two different oligomers are joined by alinker that joins the two sequences into a functional unit.

An “immobilized probe” or “immobilized nucleic acid” refers to a nucleicacid that joins, directly or indirectly, a capture oligomer to animmobilized support. A preferred immobilized probe is an oligomer joinedto a support that facilitates separation of bound target sequence fromunbound material in a sample. Supports may include known materials, suchas matrices and particles free in solution, e.g., made up ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal and preferred embodiments are magneticallyattractable particles. Preferred supports are monodisperse magneticspheres (e.g., uniform size±5%), to which an immobilized probe is joineddirectly (via covalent linkage, chelation, or ionic interaction), orindirectly (via one or more linkers), where the linkage or interactionbetween the probe and support is stable during hybridization conditions.

“Separating” or “purifying” means that one or more components of amixture, such as a sample, are removed or separated from one or moreother components. Sample components include target nucleic acids in agenerally aqueous mixture (solution phase) which may include cellularfragments, proteins, carbohydrates, lipids, and other nucleic acids.Separating or purifying removes at least 70%, preferably at least 80%,and more preferably about 95% of the target nucleic acid from othermixture components.

A “label” refers to a molecular moiety or compound that is detected orleads to a detectable signal. A label may be joined directly orindirectly to a nucleic acid probe. Direct labeling can occur throughbonds or interactions that link the label to the probe, includingcovalent bonds or non-covalent interactions, e.g. hydrogen bonds,hydrophobic and ionic interactions, or formation of chelates orcoordination complexes. Indirect labeling can occur through use of abridging moiety or linker (e.g., antibody or additional oligomer), whichis either directly or indirectly labeled, and which may amplify thedetectable signal. Labels include any detectable moiety, such as aradionuclide, ligand (e.g., biotin, avidin), enzyme, enzyme substrate,reactive group, chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Preferredlabels include a “homogeneous detectable label” that provides adetectable signal in a homogeneous reaction in which bound labeled probein a mixture exhibits a detectable change that differs from that ofunbound labeled probe, e.g., stability or differential degradation(e.g., U.S. Pat. No. 5,283,174, Arnold et al.; U.S. Pat. No. 5,656,207,Woodhead et al.; U.S. Pat. No. 5,658,737, Nelson et al.). Preferredlabels include chemiluminescent compounds, preferably acridinium ester(“AE”) compounds that include standard AE and derivatives thereof(described in U.S. Pat. Nos. 5,656,207, 5,658,737 and 5,639,604).Methods of synthesis and attaching labels to nucleic acids and detectingsignals from labels are well known (e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989), Chpt. 10; U.S. Pat. Nos.5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333).

Methods are disclosed for amplifying and detecting Legionella nucleicacid, specifically L. pneumophila 16S and 23S rRNA sequences or DNAencoding 16S and 23S rRNA. Disclosed are selected oligonucleotidesequences that specifically recognize target sequences of L. pneumophila16S and 23S rRNA or their complementary sequences, or DNA encoding 16Sand 23S rRNA. Such oligonucleotides may function as amplificationoligomers, e.g., as primers, promoter primers, blocked oligomers, andpromoter provider oligomers, whose functions are known (e.g., describedin U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 5,399,491, 5,554,516and 5,824,518, and US 2006-0046265 A1). Other embodiments may functionas probes to detect the amplified L. pneumophila sequences.

Amplification methods that use transcription mediated amplification(TMA) include the steps summarized herein (described in detail in U.S.Pat. Nos. 5,399,491, 5,554,516 and 5,824,518). The target nucleic acidthat contains the sequence to be amplified is provided as singlestranded nucleic acid (e.g., ssRNA or ssDNA) or made single stranded byconventional methods, e.g., temperature and/or chemical melting ofdouble stranded nucleic acid to provide a single-stranded target nucleicacid. A promoter primer binds specifically its target sequence in thetarget nucleic acid and an enzyme with reverse transcriptase (RT)activity extends the 3′ end of the promoter primer using the targetstrand as a template, to make a cDNA of the target sequence, which is inan RNA:DNA duplex. Enzymatic RNase activity (e.g., RNaseH) digests theRNA strand of the RNA:DNA duplex and a second primer binds specificallyto its target sequence on the cDNA strand downstream from thepromoter-primer end. The RT synthesizes a new DNA strand by extendingthe 3′ end of the second primer using the first cDNA as a template tomake a dsDNA that contains a functional promoter sequence. An RNApolymerase specific for the promoter sequence then initiatestranscription to produce multiple RNA transcripts that are, e.g., about100 to 1000 amplified copies (“amplicons”) of the initial target strandin the reaction. Amplification continues when the second primer bindsspecifically to its target sequence in each amplicon and RT makes a DNAcopy from the amplicon RNA template to produce an RNA:DNA duplex. RNasein the reaction digests the amplicon RNA from the RNA:DNA duplex and thepromoter primer binds specifically to its complementary sequence in thenewly synthesized DNA. The RT extends the 3′ end of the promoter primerto create a dsDNA that contains a functional promoter to which the RNApolymerase binds to transcribe additional amplicons that arecomplementary to the initial target strand. These autocatalyticreactions make more amplicons repeatedly during the completeamplification reaction, resulting in about a billion-fold amplificationof the target sequence that was present in the sample. The amplifiedproducts may be detected during amplification, i.e., in real-time, or atcompletion of the amplification reaction by using a probe that bindsspecifically to a target sequence in the amplified products. Signaldetected from the bound probes indicates the presence of the targetnucleic acid in the sample.

Another transcription associated amplification method summarized hereinuses one primer and one or more additional amplification oligomers toamplify nucleic acids in vitro by making transcripts (amplicons) thatindicate the presence of the target nucleic acid in a sample (describedin detail in U.S. Pat. No. 7,374,885, Becker et al.). Briefly, thissingle primer method uses a primer or “priming oligomer”, a “promoterprovider” oligomer that is modified to prevent synthetic extension fromits 3′ end (typically, by including a 3′-blocking moiety) and,optionally, a binding molecule (e.g., a 3′-blocked extender oligomer) toterminate elongation of a cDNA from the target strand. This methodincludes the steps of binding the target RNA that contains the targetsequence with a primer and, optionally, a binding molecule. The primerhybridizes to the 3′ end of the target strand and enzymatic RT activityinitiates primer extension from the 3′ end of the primer to produce acDNA, to make a duplex of the new strand and the target strand (RNA:cDNAduplex). When a binding molecule is included in the reaction, such as a3′ blocked oligomer, it binds to the target strand next to the 5′ end ofthe target sequence to be amplified. When the primer is extended by DNApolymerase activity of RT to produce the cDNA, strand, polymerizationstops when the primer extension product reaches the binding molecule onthe target strand and, thus, the 3′ end of the cDNA is determined by theposition of the binding molecule on the target strand, making the 3′ endof the cDNA complementary to the 5′ end of the target sequence. TheRNA:cDNA duplex is separated, e.g., by RNase H degradation of the RNAstrand, or by using conventional strand separation methods. Then, thepromoter provider oligomer hybridizes to the cDNA strand near its 3′end. The promoter provider oligomer includes a 5′ promoter sequence, a3′ region complementary to a sequence in the 3′ region of the cDNA, anda modified 3′ end that includes a blocking moiety to prevent initiationof DNA synthesis from the 3′ end of the promoter provider oligomer. Inthe duplex made of the promoter provider oligomer and the cDNA strand,the 3′-end of the cDNA is extended by DNA polymerase activity of the RTenzyme, using the promoter oligomer as a template to add a promotersequence to the cDNA, to make a functional double-stranded promoter. AnRNA polymerase specific for the functional promoter sequence then bindsto the promoter and transcribes RNA transcripts complementary to thecDNA which are substantially identical to the target region sequencethat was amplified from the initial target strand. The amplified RNAtranscripts then serve as substrates in the amplification process bybinding the primer and serving as a template for further cDNAproduction. This method ultimately produces many amplicons from theinitial target nucleic acid present in the sample, i.e., it makesmultiple copies of the target sequence. In embodiments of the methodthat do not include the binding molecule, the cDNA made from the primerhas an indeterminate 3′ end, but the other steps proceed as describedabove.

Detection of the amplified products may be accomplished by a variety ofmethods. The amplified nucleic acids may be associated with a surface toproduce a detectable physical change, such as an electrical signal.Amplified nucleic acids may be concentrated in or on a matrix anddetected by detecting a signal from the concentrated nucleic acid or anassociated dye (e.g., an intercalating agent such as ethidium bromide orcyber green). Nucleic acids in solution may be detected by detecting anincreased dye association in the solution phase. Preferred embodimentsdetect nucleic acid probes that are complementary to a sequence in theamplified product and form a probe:amplified product complex thatprovides a detectable signal (e.g., U.S. Pat. Nos. 5,424,413, and5,451,503, Hogan et al., and U.S. Pat. No. 5,849,481, Urdea et al.).Directly or indirectly labeled probes that specifically associate withthe amplified product provide a detectable signal to indicate thepresence of the target nucleic acid in the sample. For example, if asample contains a target nucleic acid that is L. pneumophila 16S rRNA,the amplified product contains the target sequence in or a complementarysequence of the L. pneumophila 16S rRNA, and the probe binds directly orindirectly to the amplified product's target sequence to produce asignal that indicates the presence of L. pneumophila in the sample.

Preferred probe embodiments that hybridize specifically to the amplifiedproduct sequences may be oligomers of DNA, RNA, or a mixture of DNA andRNA nucleotides, which may be synthesized with a modified backbone,e.g., a synthetic oligonucleotide that includes one or more 2′-methoxysubstituted RNA groups. Probes for detection of amplified LegionellarRNA sequences may be unlabeled and detected indirectly (e.g., bybinding to of another binding partner that is detected) or may belabeled with a label that results in a detectable signal. Preferredembodiments include label compounds that emit a detectable light signal,e.g., fluorophores or luminescent compounds detected in a homogeneousmixture. A probe may include more than one label and/or more than onetype of label, or detection may rely on using a mixture of probes inwhich each probe is labeled with a compound that produces a detectablesignal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579). Labels may beattached to a probe by any of a variety of known means, e.g., covalentlinkages, chelation, and ionic interactions, but preferred embodimentscovalently link the label to the oligonucleotide. Probes may besubstantially linear oligonucleotides, i.e., lacking conformations heldby intramolecular bonds, or may be include functional conformationalstructures, i.e., conformations such those found in hairpin structureprobes held together by intramolecular hybridization. Preferredembodiments of linear oligomers generally include a chemiluminescentlabel, preferably an AE compound.

Hairpin probes are preferably labeled with any of a variety of differenttypes of interacting labels, where one interacting member is usuallyattached to the 5′ end of the hairpin probe and the other interactingmember is attached to the 3′ end of the hairpin probe. Such interactingmembers, which may be generally referred to as a reporter dye and aquencher, include a luminescent/quencher pair, luminescent/adduct pair,Forrester energy transfer pair, or a dye dimer. A luminescent/quencherpair may be made up of one or more luminescent labels, such aschemiluminescent or fluorescent labels, and one or more quenchers. Inpreferred embodiments, a hairpin probe is labeled at one end with afluorescent label (“F”) that absorbs light of a particular wavelength orrange and emits light another emission wavelength or range and at theother end with a quencher (“Q”) that dampens, partially or completely,signal emitted from the excited F when Q is in proximity with thefluorophore. Such a hairpin probe may be referred to as labeled with afluorescent/quencher (F/Q) pair. Fluorophores are well known compoundsthat include, e.g., acridine, fluorescein, sulforhodamine 101,rhodamine, 5-(2′-aminoethyl)aminoaphthaline-1-sulfonic acid (EDANS),TEXAS RED®, Eosine, BODIPY® and lucifer yellow (Tyagi et al., NatureBiotechnology 16:49-53, 1998). Quenchers are also well known andinclude, e.g., 4-(4′-dimethyl-amino-phenylaxo)benzoic acid (DABCYL),thallium, cesium, and p-xylene-bis-pyridinium bromide. Different F/Qcombinations are well known and many combinations may function together,e.g., DABCYL with fluorescein, rhodamine, or EDANS. Other combinationsof labels for hairpin probes include a reporter dye, e.g., FAM™, TET™,JOE™, VIC™ combined with a quencher such as TAMRA™ or a non-fluorescentquencher.

A preferred embodiment of a hairpin probe is a “molecular torch” thatdetects an amplified product to indicate the presence of a targetLegionella sequence in a sample after the amplification step. Amolecular torch includes: (1) a target detection means that hybridizesto the target sequence, resulting in an open conformation; (2) a torchclosing means that hybridizes to the target detecting means in theabsence of the target sequence, resulting in a closed conformation; and(3) a joining means that joins the target detection means and the torchclosing means (described in detail in U.S. Pat. Nos. 6,849,412,6,835,542, 6,534,274, and 6,361,945). A torch probe in open conformationresults in a detectable signal that indicates the presence of theamplified target sequence, whereas the closed conformation produces anamount of signal that is distinguishable from that of the openconformation indicating that the target sequence is not present. Anotherpreferred hairpin probe embodiment is a “molecular beacon” that includesa label on one arm of the hairpin sequence, a quencher on the other arm,and a loop region joining the two arms (described in detail in U.S. Pat.Nos. 5,118,801 and 5,312,728). Methods for using such hairpin probes arewell known in the art.

Oligomers that are not extended by a nucleic acid polymerase include ablocker group that replaces the 3′ OH to prevent enzyme-mediatedextension of the oligomer in an amplification reaction. Blockedamplification oligomers and/or blocked detection probes present duringamplification (for real time detection) preferably lack a 3′ OH butinclude one or more blocking groups located at or near the 3′ end. Ablocking group is covalently attached to the 3′ terminus of theoligonucleotide or is located near the 3′ end, preferably within fiveresidues of the 3′ end, and is sufficiently large to limit binding of apolymerase to the oligomer. Many different chemical groups may be usedas a blocking moiety, e.g., alkyl groups, non-nucleotide linkers,alkane-diol dideoxynucleotides, and cordycepin.

A preferred method for detection of L. pneumophila sequences uses atranscription-associated amplification with a hairpin probe, e.g.molecular torch or molecular beacon, because the probe may be addedbefore amplification, and detection is carried out without furtheraddition of reagents. For example, a probe may be designed so that theTm of the hybridized arms of the hairpin probe (e.g., target bindingdomain:target closing domain complex of a molecular torch) is higherthan the amplification reaction temperature to prevent the probe fromprematurely binding to amplified target sequences. After an interval ofamplification, the mixture is heated to open the torch probe arms andallow the target binding domain to hybridize to its target sequence inthe amplified product. The solution is then cooled to close probes notbound to amplified products, which closes the label/quencher (F/Q) pair,allowing detection of signals from probes hybridized to the amplifiedtarget sequences in a homogeneous reaction. For example, the mixturecontaining the F/Q labeled hairpin probe is irradiated with theappropriate excitation light and the emission signal is measured.

In other embodiments, the hairpin detection probe is designed so thatamplified products preferentially hybridize to the target binding domainof the probe during amplification, thereby changing the hairpin from itsclosed to open conformation as amplification progresses. Theamplification reaction mixture is irradiated at intervals during theamplification reaction to detect the emitted signal from the open probesduring amplification, i.e., in real time.

Preparation of samples for amplification of Legionella sequences mayinclude separating and/or concentrating organisms contained in a samplefrom other sample components, e.g., filtration of particulate matterfrom air, water or other types of samples. Sample preparation may alsoinclude chemical, mechanical, and/or enzymatic disruption of cells torelease intracellular contents, including Legionella 16S or 23S rRNA orDNA encoding the 16S or 23S rRNA. Sample preparation may include a stepof target capture to specifically or non-specifically separate thetarget nucleic acids from other sample components. Nonspecific targetpreparation methods may selectively precipitate nucleic acids from asubstantially aqueous mixture, adhere nucleic acids to a support that iswashed to remove other sample components, or use other means tophysically separate nucleic acids, including Legionella nucleic acid,from a mixture that contains other components. Other nonspecific targetpreparation methods may selectively separate RNA, including Legionella16S or 23S rRNA, from DNA in a sample.

In a preferred embodiment, Legionella rRNA or DNA encoding rRNA areselectively separated from other sample components by specificallyhybridizing the Legionella nucleic acid to a capture oligomer specificfor the Legionella target sequence to form a target sequence:captureprobe complex that is separated from sample components. A preferredembodiment of specific target capture binds the Legionellatarget:capture probe complex to an immobilized probe to form atarget:capture probe:immobilized probe complex that is separated fromthe sample and, optionally, washed to remove non-target samplecomponents. The capture probe includes a sequence that specificallybinds to the Legionella target sequence in 16S or 23S rRNA or in DNAencoding 16S or 23S rRNA and also includes a specific binding partnerthat attaches the capture probe with its bound target sequence to asupport (e.g., matrix or particle), which facilitates separating thetarget sequence from the sample components. In a preferred embodiment,the specific binding partner of the capture probe is a 3′ tail sequencethat is not complementary to the Legionella target sequence but thathybridizes to a complementary sequence on an immobilized probe attachedto the support. Preferred 3′ tail sequences are substantiallyhomopolymeric 10 to 40 nt sequences (e.g., A₁₀ to A₄₀) that bind to acomplementary immobilized sequence (e.g., poly-T) attached to thesupport. Target capture occurs in a solution phase mixture that containscapture oligomers that hybridize specifically to the Legionella targetnucleic acid under hybridizing conditions, usually at a temperaturehigher than the Tm of the tail sequence:immobilized probe sequenceduplex. The Legionella target:capture probe complex is captured byadjusting the hybridization conditions so that the capture probe tailthen hybridizes to the immobilized probe, and the entire complex on thesupport is separated from the other sample components. The support withthe attached complex that includes the Legionella target sequence may bewashed to further remove other sample components. Preferred supports areparticulate, such as paramagnetic beads, so that particles with thecomplex that includes the captured Legionella target sequence may besuspended in a washing solution and retrieved from the washing solutionby using magnetic attraction. In other embodiments, the capture probemay bind nonspecifically to nucleic acids in the sample, including theLegionella target sequence, and then similar steps of attaching thecapture probe:nucleic acid complexes to a support and separating thecaptured complexes on the support are performed. Whether target captureis specific or non-specific for the Legionella target sequence, thecaptured nucleic acids are then subjected to in vitro amplificationspecific for the intended Legionella target sequence. To limit thenumber of handling steps, Legionella target nucleic acid may beamplified by mixing the Legionella target sequence in the capturedcomplex on the support with amplification reagents, or a primer may beincluded in the target capture reaction mixture, thus allowing theLegionella specific primer and target sequences to hybridize duringtarget capture and be separated together from the sample in the capturedcomplex.

Assays for detection of Legionella nucleic acid may optionally include anon-Legionella internal control (IC) nucleic acid that is amplified anddetected in the same assay reaction mixtures by using amplification anddetection oligomers specific for the IC sequence. Amplification anddetection of a signal from the amplified IC sequence demonstrates thatthe assay reagents, conditions, and procedural steps were properly usedand performed in the assay if no signal is obtained for the intendedtarget Legionella nucleic acid (e.g., samples that provide negativeresults for L. pneumophila). The IC may be used as an internalcalibrator for the assay when a quantitative result is desired, i.e.,the signal obtained from the IC amplification and detection is used toset a parameter used in an algorithm for quantitating the amount ofLegionella nucleic acid in a sample based on the signal obtained foramplified an Legionella target sequence. A preferred IC embodiment is arandomized sequence that has been derived from a naturally occurringsource (e.g., an HIV sequence that has been rearranged in a randommanner). A preferred IC may be an RNA transcript isolated from anaturally occurring source or synthesized in vitro, such as by makingtranscripts from a cloned randomized sequence such that the number ofcopies of IC included in an assay may be accurately determined. Theprimers and probe for the IC target sequence are designed andsynthesized by using any well known method provided that the primers andprobe function for amplification of the IC target sequence and detectionof the amplified IC sequence using substantially the same assayconditions used to amplify and detect the Legionella target sequence andthe IC components in the assay do not interfere with those used toamplify and detect the Legionella target sequence. In preferredembodiments that include a target capture-based purification step, atarget capture probe specific for the IC target is included in thetarget capture step so that the IC is treated in the same conditions asused for the intended Legionella analyte in all of the assay steps.

Amplification and Detection of 16S rRNA Sequences of L. pneumophila

For amplification and detection of target sequences in 16S rRNAsequences (which include 16S rRNA and DNA encoding 16S rRNA) of L.pneumophila, oligomers were designed that act as amplification oligomersand detection probes by comparing known sequences of 16S rRNA or genesequences encoding 16S rRNA and selecting sequences that are common toL. pneumophila isolates, but preferably are not completely identical to16S rRNA sequences of other Legionella species or other bacteria.Sequence comparisons were conducted by using known 16S rRNA sequences(rRNA or genes) of Legionella species (L. anisa, L. beliardensis, L.briminghamiensis, L. bozemanii, L. brunensis, L. busanensis, L. cherrii,L. cincinatiensis, L. dumoffii, L. erythra, L. feeleii, L. gresilensis,L. gratiana, L. hackeliae, L. jamestowniensis, L. jordansis, L. lyticum,L. longbeachae, L. micdadei, L. moravica, L. oakridgenesis, L.parisiensis, L. pneumophila, L. quateirensis, L. rubrilucens, L.santicrucis, L. sainthelensi, L. shakespearei, L. spiritensis, L.steigerwaltii, L. taurinesis, L. wadsworthii) and of other bacterialspecies (Escherichia coli, Pseudomonas aeruginosa, P. alcaligenes, P.stutzeri, P. putida, P. syringae, Bordetella parapertus, B.bronchiseptica, Corynebacterium xerosis, C. pseudotuberculosis,Klebsiella pneumoniae, and Haemophilus influenzae). Specificoligonucleotide sequences were selected, synthesized in vitro, and theoligonucleotides were characterized by determining their Tm andhybridization characteristics with complementary target sequences(synthetic or purified rRNA sequences from bacteria) by using standardlaboratory methods. The selected oligomers were further tested by usingdifferent combinations of the amplification oligomers in amplificationreactions with templates that were synthetic 16S RNA target sequences or16S rRNA purified from various Legionella species grown in culture, todetermine the relative efficiencies of amplification of the targetsequences by using the selected amplification oligomers. Theefficiencies of different combinations of oligomers were monitored bydetecting the amplified products of the amplification reactions,generally by binding a labeled probe oligomer to the amplified productsand detecting the relative amount of signal that indicated the amount ofamplified product. Generally, for initial testing of amplificationefficiency, linear detection probes labeled with an AE compound werehybridized to the amplified products and detected by using ahybridization protection assay (HPA) that selectively degrades the AElabel in unhybridized probes and detects the signal from hybridizedprobes (substantially as described in U.S. Pat. Nos. 5,283,174,5,656,207, 5,658,737 and 5,824,475).

Preferred embodiments of the selected amplification oligomers for L.pneumophila 16S rRNA target sequences are shown in Table 1.Amplification oligomers include those that may function as primers,promoter primers, and/or promoter provider oligomers. For the lattertwo, promoter sequences are shown in lower case in Table 1. Someoligomer embodiments include only the target-specific sequence of acorresponding promoter primer or promoter provider oligomer, e.g., SEQID NO:30 is a target-specific sequence that is identical to thetarget-specific sequence contained in SEQ ID NO:31, which includes a 5′promoter sequence. Those skilled in the art will appreciate that thetarget-specific sequences listed in Table 1 may optionally be attachedto the 3′ end of any known promoter sequence to function as a promoterprimer or promoter provider with the appropriate RNA polymerase for thechosen promoter sequence. Preferred embodiments include a promotersequence specific for the RNA polymerase of bacteriophage T7 (e.g., SEQID Nos. 90, 91, or 92). Preferred embodiments of amplification oligomersmay include a mixture of DNA and RNA bases, and 2′ methoxy RNA groups,e.g., oligomers of SEQ ID Nos. 56 and 57 may include RNA bases and 2′methoxy linkages at the first four positions from the 5′ end.Embodiments of amplification oligomers may be modified by synthesizingthe oligomer with a 3′ blocked to make the oligomer optimal forfunctioning as a blocking molecule or promoter provider oligomer in asingle primer transcription associated amplification reaction. Preferredembodiments of 3′ blocked oligomers include those of SEQ ID Nos. 58, 59,60 and 61 that include a blocked C near or at the 3′ end.

TABLE 1 Amplification Oligomers for Amplificationof Legionella 16S rRNA Target Sequences SEQ Sequence ID NO.GAGAGGGTAGTGGAATTTCCG 28 GTAGAGATCGGAAGGAACACCAG 29 TGTTTGCTCCCCACGCTT30 aatttaatacgactcactatagggagaTGTTTGCTCCCCACGC 31TTCCAGGGTATCTAATCCTGTTTGCTC 32aatttaatacgactcactatagggagaCCAGGGTATCTAATCC 33 TGTTTGCTCCCATGCAGCACCTGTATCAG 34 aatttaatacgactcactatagggagaCCATGCAGCACCTGTA 35TCAG GCCATGCAGCACCTGTAT 36 aatttaatacgactcactatagggagaGCCATGCAGCACCTGT37 AT GATTAAAACTCAAAGGAATTGACGGGG 38 AAGCGGTGGAGCATGTGG 39CTACCCTCTCCCATACTCGAG 40 aatttaatacgactcactatagggagaCTACCCTCTCCCATAC 41TCGAG GAGTTGCAGACTCCAATCCG 42aatttaatacgactcactatagggagaGAGTTGCAGACTCCAA 43 TCCGGAGTCGAGTTGCAGACTCCAATC 44 aatttaatacgactcactatagggagaGAGTCGAGTTGCAGAC45 TCCAATC GTAATACGGAGGGTGCGAG 46 CGCCCTCTGTATCGGCCATTGTAGC 47CCAGGTCGCCCCTTCGC 48 CCAATCCGGACTACGAACGGCTTTTGAGGATTGGCT 49CCAATCCGGACTACGACCGACTTTTAAGGATTTGCT 50 GGATGACGTCAAGTCATCATGG 51CTTACGGGTAGGGCTACACACGTG 52 GCTACACCGGAAATTCCACTAC 53aatttaatacgactcactatagggagaGCTACACCGGAAATTC 54 CACTACCGAGCGTTAATCGGAATTACTGG 55 GCUACACCGGAAATTCCACTAC 56CGGAAATTCCACTACCCTCTCC 57 CUUUACGCCCAGUAAUUCCG 58 GCUGGCACGCUCCGUAUUAC59 aatttaatacgactcactatagggagaCGTAAAGGGTGCGTAG 60 GTGGTTGaatttaatacgactcactatagggagaCGAGCGTTAATCGGAA 61 TTACTGG

Preferred embodiments of the selected detection probes for detectingamplified products of 16S rRNA sequences or DNA encoding 16S rRNA areshown in Table 2. Preferred embodiments of linear detection probes arelabeled with a chemiluminescent AE compound attached to the probeoligomer via a linker (substantially as described in U.S. Pat. Nos.5,585,481, and 5,639,604, particularly at column 10, line 6 to column11, line 3, and Example 8). Examples of preferred labeling positions area central region of the probe oligomer and near a region of A:T basepairing, at a 3′ or 5′ terminus of the oligomer, and at or near amismatch site with a known sequence that is not the desired targetsequence. Preferred embodiments of such AE-labeled oligomers includethose with a linker between: nt 4 and nt 5 of SEQ ID NO:6, nt 5 and nt 6of SEQ ID Nos. 2 and 14, nt 6 and nt 7 of SEQ ID NO:14, nt 7 and nt 8 ofSEQ ID Nos. 7 and 18, nt 8 and nt 9 of SEQ ID Nos. 13 and 14, nt 9 andnt 10 of SEQ ID Nos. 2, 11, 24, 26, and 27, nt 10 and nt 11 of SEQ IDNos. 4, 12, 15, and 16, nt 11 and nt 12 of SEQ ID NO:25, nt 12 and nt 13of SEQ ID Nos. 7 and 18, nt 13 and nt 14 of SEQ ID Nos. 7, 10, 17, and23, nt 14 and nt 15 of SEQ ID Nos. 1, 2, 3, 16, 21, and 22, nt 15 and nt16 of SEQ ID Nos. 9 and 15, nt 16 and nt 17 of SEQ ID Nos. 5, 8, 13, and14, nt 17 and nt 18 of SEQ ID NO:11, nt 18 and nt 19 of SEQ ID NO:6, andnt 19 and nt 20 of SEQ ID Nos. 10, 13, and 14. Detection probes may beused with one or more helper probes that are unlabeled and facilitatebinding of the labeled detection probe to its target (U.S. Pat. No.5,030,557, Hogan et al.). Preferred embodiments of helper probes includethose of SEQ ID Nos. 19 and 20. Other detection probe embodiments areoligomers that form hairpin configurations by intramolecularhybridization of the probe sequence. Preferred embodiments of hairpinprobe oligomers include the molecular torches of SEQ ID Nos. 62, 63, 64,and 65 in Table 2, in which lower case letters are used for the torchclosing means that hybridize to the torch's target detecting means (i.e.the target-complementary region) in the absence of the target sequence.The target-complementary regions of SEQ ID Nos. 62, 63, 64, and 65 areprovided in Table 2 as SEQ ID Nos. 93, 94, 95 and 96, respectively.Preferred hairpin probe oligomers are synthesized with a fluorescentlabel attached at one end and a quencher compound attached at the otherend of the sequence. Embodiments of hairpin probes may be labeled with a5′ fluorophore and a 3′ quencher, e.g., 5′ fluorescein label with 3′DABCYL quencher. Some embodiments of hairpin oligomers include anon-nucleotide linker moiety at selected positions within the sequence,e.g., oligomers that include an abasic 9-carbon (“C9”) linker locatedin: SEQ ID NO:62 between nt 5 and nt 6 or nt 20 and nt 21, SEQ ID NO:63between nt 5 and nt 6 or nt 23 and nt 24, SEQ ID NO:64 between nt 23 andnt 24, and SEQ ID NO:65 between nt 25 and nt 26. Preferred embodimentsof detection probe oligomers may also include a mixture of DNA and RNAbases, and 2′ methoxy RNA groups. 2′ methoxy modified RNA probeoligomers are exemplified by SEQ ID Nos. 14, 62, 63, 64 and 65.

TABLE 2 Probes for Detection of Amplified Sequencesof Legionella 16S rRNA Target Sequences SEQ Sequence ID NO.GTATTAGGCCAGGTAGCCG  1 CGGCTACCTGGCCTAATAC  2 TGGCGAAGGCGGCTACCTGG  3GAAGGCGGCTACCTGGCCTAATACTG  4 GGCGGCTACCTGGCCTAATACTGACAC  5CTGTAAACGATGTCAACTAGCTGTTGG  6 CTTACCTACCCTTGACATACAGTG  7CAACGCGAAGAACCTTACCTACCCTTGACATAC  8 CGAAGAACCTTACCTACCCTTGACATACAGTG  9CCTTACCTACCCTTGACATACAGTGAATTTTGCAGAGATG 10 GCTTAACCTGGGACGGTCAGATAATAC11 TTAACCTGGGACGGTCAGATAAT 12 CCTGGGACGGTCAGATAATACTGGTTG 13CCUGGGACGGUCAGAUAAUACUGGUUG 14 CTGGGACGGTCAGATAATACTGGTTG 15TGGGACGGTCAGATAATACTGGTTG 16 GGGACGGTCAGATAATACTGGTTGAC 17GGACGGTCAGATAATACTGGTTG 18 CTACAATGGCCGATACAGAGGGCGGC 21CGTAAAGGGTGCGTAGGTGGTTGATTAAG 22 GTAAAGGGTGCGTAGGTGGTTGATT 23GATTAAGTTATCTGTGAAATTCCTGG 24 CGCGTAGGAATATGCCTTGAAG 25GGCCTGGCGCTTTAAGATTAGC 26 CGGCUACCUGGCCUAAUAC 27 AACAGUAUUAUCUGACCGUCCC93 gggACCAGUAUUAUCUGACCGUCCC 62 CAACCAGUAUUAUCUGACCGUCC 94ggacgCAACCAGUAUUAUCUGACCGUCC 63 CAACCAGUAUUAUCUGACCGUC 95cCAACCAGUAUUAUCUGACCGUCgguugg 64 GUCAACCAGUAUUAUCUGACCGUC 96cGUCAACCAGUAUUAUCUGACCGUCgacg 65

Embodiments of capture probe oligomers for use in sample preparation toseparate Legionella 16S rRNA target nucleic acids from other samplecomponents include those that contain the target-specific sequences ofSEQ ID NO: 66 (GCTGCCGTTCGACTTGCATGTG), SEQ ID NO:67(ATCGTCGCCTTGGTAGGCCC), and SEQ ID NO:68 (GCCGGTGCTTCTTCTGTGGGTAACG).Preferred embodiments of the capture probes include a 3′ tail regioncovalently attached to the target-specific sequence to serve as abinding partner that binds a hybridization complex made up of the targetnucleic acid and the capture probe to an immobilized probe on a support.Preferred embodiments of capture probes that include the target-specificsequences of SEQ ID Nos. 66, 67,and 68, further include 3′ tail regionsmade up of substantially homopolymeric sequences, e.g., a dT₃A₃₀sequence.

Reagents used in target capture, amplification and detection stepsdescribed in the examples herein generally include one or more of thefollowing. Sample Transport Solution: 15 mM sodium phosphate monobasic,15 mM sodium phosphate dibasic, 1 mM EDTA, 1 mM EGTA, and 3% (w/v)lithium lauryl sulfate (LLS), pH 6.7. Lysis buffer: 790 mM HEPES, 230 mMsuccinic acid, 10% (w/v) LLS, and 680 mM LiOH. Specimen Dilution Buffer:300 mM HEPES, 3% (w/v) LLS, 44 mM LiCl, 120 mM LiOH, 40 mM EDTA, pH 7.4.Target Capture Reagent: 250 mM HEPES, 310 mM LiOH, 1.88 M LiCl, 100 mMEDTA, pH 6.4, and 250 μg/ml of paramagnetic particles (0.7-1.05μparticles, SERA-MAO MG-CM, Seradyn, Inc., Indianapolis, Ind.) withcovalently bound (dT)₁₄ oligomers. Wash Solution: (for target capture)10 mM HEPES, 150 mM NaCl, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol,0.02% (w/v) methyl paraben, 0.01% (w/v) propyl paraben, and 0.1% (w/v)sodium lauryl sulfate, pH 7.5. Amplification reagent: a concentratedmixture that was mixed with other reaction components (e.g., sample orspecimen dilution buffer) to produce a mixture containing 47.6 mMNa-HEPES, 12.5 mM-acetyl-L-cysteine, 2.5% TRITON™ X-100, 54.8 mM KCl, 23mM MgCl₂, 3 mM NaOH, 0.35 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 7.06mM rATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85 mM rGTP, 0.26 mM Na₂EDTA, 5%v/v glycerol, 2.9% trehalose, 0.225% ethanol, 0.075% methylparaben,0.015% propylparaben, and 0.002% Phenol Red, pH 7.5-7.6. Amplificationoligomers (primers, promoter primers, blocker oligomers, or promoterprovider oligomers), and optionally probes, may be added to the reactionmixture in the amplification reagent or separately. Enzymes in TMAreactions: about 90 U/μl of MMLV reverse transcriptase (MMLV-RT) andabout 20 U/μl of T7 RNA polymerase per reaction (1 U of RT incorporates1 nmol of dTTP in 10 min at 37° C. using 200-400 μM oligo dT-primedpolyA template, and 1 U of T7 RNA polymerase incorporates 1 nmol of ATPinto RNA in 1 hr at 37° C. using a T7 promoter in a DNA template). ProbeReagent: for AE-labeled detection probes was 100 mM lithium succinate,0.1% to 3% (w/v) LLS, 10 mM mercaptoethanesulfonate, and optionally 3%(w/v) polyvinylpyrrolidone. Hybridization Reagent: for AE-labeled probebinding to nucleic acid was 100 mM succinic acid, 2% (w/v) LLS, 100 mMLiOH, 15 mM aldrithiol-2, 1.2 M LiCl, 20 mM EDTA, and 3.0% (v/v)ethanol, pH 4.7. Selection Reagent for preferentially hydrolyzing an AElabel on unbound detection probes was 600 mM boric acid, 182.5 mM NaOH,1% (v/v) octoxynol (TRITON® X-100), pH 8.5. Detection Reagents forproducing a chemiluminescent response from AE labels comprised DetectReagent 1 (1 mM nitric acid and 32 mM H₂O₂), and Detect Reagent II (1.5M NaOH) to neutralize the pH (U.S. Pat. Nos. 5,283,174, 5,656,744, and5,658,737). All of the reagent addition and mixing steps may beperformed manually, or by using a combination of manual and automatedsteps, or by using a completely automated system. Amplification methodsthat use TMA use procedures substantially as disclosed in U.S. Pat. Nos.5,399,491 and 5,554,516. Amplification methods that use single primertranscription associated amplification use procedures substantially asdisclosed in U.S. Pat. No. 7,374,885, Becker et al. Use of AE-labeledprobes and signal detection to detect hybridization complexes withtarget sequences use procedures substantially as disclosed in U.S. Pat.Nos. 5,283,174, 5,656,744, and 5,658,737. Methods for using hairpinprobes have been disclosed in detail in U.S. Pat. Nos. 6,849,412,6,835,542, 6,534,274, and 6,361,945.

By using various combinations of these amplification oligomers andAE-labeled detection probes to provide a detectable chemiluminescentsignal, L. pneumophila 16S rRNA sequences were specifically detectedwhen the sample contained about 100 copies of the 16S rRNA targetsequence. Preferred embodiments of the methods are illustrated inExamples 1 to 4.

Example 1: Specific Amplification and Detection of L. pneumophila TargetSequence

Known numbers of in vitro transcripts of 16S rRNA sequences from L.pneumophila and L. wadsworthii were separately amplified in TMAreactions using the same conditions and combinations of amplificationoligomers and the amplified products were detected by using the sameprobe (SEQ ID NO:13 labeled with AE between nt 16 and 17). Briefly,specimens were prepared by mixing in specimen dilution buffer 100, 1000,10000, or 100000 copies of L. pneumophila or L. wadsworthii 16S rRNA invitro transcripts. Specimens were mixed with amplification reagent andamplification oligomers (combinations of amplification oligomers of SEQID Nos. 41 and 46, or 41 and 55, or 54 and 46, or 54 and 55) andamplified in TMA reactions using substantially the procedures describedin detail in U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacian et al.Briefly, the reaction mixture (about 0.08 ml) containing amplificationreagent, target nucleic acid, and amplification oligomers (15 pmol ofeach oligomer in a combination per reaction) was mixed, covered withsilicon oil (0.2 ml) to prevent evaporation, and incubated for 10 min at62° C. and then for 5 min at 42° C. Then the enzyme reagent (0.025 mlcontaining MMLV-RT and T7 RNA polymerase) was added, and the reactionmixtures were incubated for 60 min at 42° C. Following amplification,detection of the amplified products involved mixing the amplificationmixture with a labeled detection probe oligomer of SEQ ID NO:13 (0.1pmol per reaction in 0.1 ml of probe reagent, an amount previouslydetermined to produce a maximum detectable signal in a detection rangeof about 5,000,000 relative light units (“RLU”)) from the hybridizedlabeled probe). The mixtures of probe and amplified sequences wereincubated to allow the detection probe to hybridize to the amplifiedproduct and the chemiluminescent signal produced from hybridized probeswas detected substantially as described in U.S. Pat. Nos. 5,283,174 and5,639,604. Briefly, the probe and amplified product mixtures wereincubated for 20 min at 62° C., then cooled at room temperature forabout 5 min, and selection reagent (0.25 ml) was added, mixed, andincubated 10 min at 62° C. followed by room temperature for 15 min tohydrolyze the label on unbound probes. Chemiluminescent signal from AEon bound probes was produced by adding Detect Reagent I, incubation, andadding Detect Reagent II, and signal (RLU) was measured by using aluminometer (e.g., LEADER®, Gen-Probe Incorporated, San Diego, Calif.).Results of these assays are shown in Table 3 as the average RLU for twoassays performed for each of the amplification oligomers combinations.In all cases, negative controls (i.e., reactions performed in the samemanner using specimens with 0 copies of target) provided a backgroundsignal of between 1996 and 2579 RLU. The results show that theamplification oligomer combinations of SEQ ID NOS. 41 and 46, or 41 and55, or 46 and 54, or 55 and 54 in a TMA reaction were able to amplify asfew as 100 copies of 16S rRNA target sequence from L. pneumophila, asdetected by the signal from AE-labeled probe of SEQ ID NO:13 hybridizedto amplified products. In contrast, using the same conditions andcombinations of amplification oligomers and detection probe, producedgenerally negative results when L. wadsworthii 16S rRNA was used. Onlyreactions that included high copy numbers of L. wadsworthii 16S rRNA(10000 to 100000) produced a signal above the background.

TABLE 3 Assays Using 16S rRNA Sequences from L. pneumophila and L.wadsworthii Amplification Oligomers (SEQ ID NOS) Target Source Copies41 + 46 41 + 55 54 + 46 54 + 55 L. pnuemophila 100 69,967 399,7681,625,390 969,753 L. pnuemophila 1000 778,085 1,319,282 4,769,7163,995,973 L. pnuemophila 10000 4,859,408 4,681,879 4,928,583 4,892,200L. pnuemophila 100000 5,057,675 4,952,711 5,016,147 4,812,010 L.wadsworthii 100 2,120 2,418 2,385 2,155 L. wadsworthii 1000 2,317 3,1823,916 2,389 L. wadsworthii 10000 3,253 20,540 4,292 3,545 L. wadsworthii100000 10,766 160,667 24,577 14,264

Similar assays were performed using the amplification oligomercombinations of SEQ ID Nos. 51 and 43, or 52 and 43, or 51 and 45, or 52and 45 (each primer used at 15 pmol per reaction) and an AE-labeleddetection probe of SEQ ID NO:21. TMA amplification and chemiluminescentdetection steps were performed substantially as described above, usingin vitro transcripts of 16S rRNA sequences of L. pneumophila or L.wadsworthii as the target nucleic acid, at 10, 100, 1000, 10000, and100000 copies per reaction. Amplification oligomer combinations of SEQID Nos. 52 and 43 reliably detected 10000 and 100000 copies of L.pneumophila target (average RLU of 315, 437 and 2,206,102, respectively)and did not detect the same number of copies of L. wadsworthii target(average RLU of 7,152 and 7,437, respectively). The amplificationoligomer combination of SEQ ID Nos. 52 and 45 reliably detected 1000,10000 and 100000 copies of L. pneumophila target (average RLU of148,869, 874,748, and 4,099,682, respectively) and did not detect thesame number of copies of L. wadsworthii target (average RLU of 12,045,7,868, and 20,482, respectively). The other amplification oligomercombinations did not reliably provide a positive signal for the L.pneumophila target sequence.

Example 2: Specific Amplification and Detection of L. pneumophila TargetSequences

Using the procedures substantially as described in Example 1, similarTMA reactions were performed by using amplification oligomers of SEQ IDNos. 54 and 55 (15 pmol each per reaction), and an AE-labeled detectionprobe of SEQ ID NO:13 (100 fmol per reaction), using purified extractsof total RNA from cultured bacteria that were three strains of L.pneumophila, other Legionella species (L. longbeachae, L. micdadei, L.spiritensis, and L. wadsworthii), and non-Legionella species (K.pneumoniae, B. parapertussis, and B. bronchiseptica) as the targetnucleic acids. Total RNA were purified using standard methods toreversibly bind RNA to a support (e.g., as described by the manufacturerfor an AMBI ON® RNAqueous product) and purified RNA were quantitated byusing standard fluorometry methods. Each RNA target was testedindividually for each source in assays using 4 replicate samples.Positive controls were amplified using the same conditions but usingknown amounts of L. pneumophila in vitro transcripts of 16S rRNAsequences, as described in Example 1. Positive controls were assayedusing duplicate reactions that detected 100 or more copies per reaction(average RLU detected were: 1,504,998 RLU for 100 copies, 4,754,776 for1000 copies, 5,136,953 for 10000 copies, and 5,267,713 for 100000copies). Negative controls were reaction mixtures that contained notarget nucleic acid which provided background signals of 2041 and 2095RLU. The results (average RLU) of these assays for the total RNA fromdifferent targets are shown in Table 4. The results show that the methodspecifically amplifies and detects L. pneumophila 16S rRNA targetsequences and does not significantly amplify and/or detect other 16SrRNA sequences from other Legionella species or common non-Legionellabacteria. The assay detected signal from amplified 16S rRNA target from10 fg/reaction of L. pneumophila purified rRNA, but the same conditionsprovided negative results when 10 fg/reaction or 100 fg/reaction ofpurified rRNA were tested from L. longbeachae, L. micdadei, L.spiritensis, L. wadsworthii, K. pneumoniae, B. parapertussis, and B.bronchiseptica sources.

TABLE 4 Detection Assays Using RNA Isolated from Different BacterialSources Amount Target Source 10 fg per reaction 100 fg per reaction L.pneumophila (strain 1) 5,074,970 5,203,724 L. pneumophila (strain 2)4,993,084 5,257,040 L. pneumophila (strain 3) 5,027,967 5,267,307 L.longbeachae 2,752 3,453 L. micdadei 1,871 2,105 L. spiritensis 1,8791,908 L. wadsworthii 10,939 82,230 K. pneumoniae 1,934 1,890 B.parapertussis 1,808 1,657 B. bronchiseptica 1,914 1,767

Example 3: Amplification and Detection of L. pneumophila 16S rRNA TargetSequence

TMA reactions were performed substantially as described in Example 1,but using different amplification oligomer combinations: SEQ ID Nos. 29and 31, or 28 and 31, or 29 and 33, or 28 and 33 (15 pmol each perreaction), using L. pneumophila 16S rRNA in vitro transcripts as thetarget for amplification. Amplified products were detected substantiallyas described in Example 1, but using an AE-labeled probe of SEQ ID NO:5.All of the amplification oligomer combinations detected 1000 or morecopies of the target nucleic acid, and may have detected fewer copies ofthe target but the signals were obscured by relatively high backgrounddetected in the negative control tests (without added target nucleicacid). Of these combinations, oligomers of SEQ ID Nos. 29 and 31, andSEQ ID Nos. 28 and 31 performed best in the assays that detected L.pneumophila 16S rRNA sequences.

Example 4: Target Capture, Amplification and Detection of L. pneumophila16S rRNA Sequence

This methods presented here included purification of the target nucleicacid from a sample before the amplification step. Target purificationwas done by using target capture, substantially as described in U.S.Pat. Nos. 6,110,678, 6,280,952, and 6,534,273. Briefly, samples wereprepared containing known amounts of 16S rRNA target nucleic acid (invitro transcripts at 1, 10, 100 and 10000 copies per sample in a totalvolume of 0.4 ml of sample transport solution), and mixed with a targetcapture oligomer (2.5 pmol per assay) of SEQ ID NO:66 or SEQ ID NO:67,to which dT₃A₃₀ tails had been covalently attached, and magneticparticles with covalently attached polydT oligomers. The mixtures wereincubated first for 30 min at 60° C., then for 30 min at roomtemperature to form hybridization complexes that captured Legionella RNAto the particles. Magnetic particles with captured Legionella RNA wereseparated by applying a magnetic field for 10 min to the containerexterior, then the solution phase was aspirated away to remove othersample components, and the particles with attached hybridizationcomplexes were washed twice sequentially (each with 1 ml of washsolution at room temperature, aspirating the wash solution away from themagnetized particles). The particles with attached hybridizationcomplexes including the Legionella target nucleic acid were suspended inamplification reagent containing amplification oligomers of SEQ ID Nos.54 and 55 (each at 15 pmol per assay), and TMA reactions were performedsubstantially as described in Example 1. Then, the amplified productswere detected by using an AE-labeled probe of SEQ ID NO:15 (0.1 pmol perassay) and the chemiluminescent signals were detected substantially asdescribed in Example 1. Duplicate samples were prepared and assayed foreach condition and the results, reported as average detected RLU, areshown in Table 5. Negative controls were treated identically butcontained no target RNA, and provided backgrounds in a range of 1485 to1640 RLU. The results in Table 5 show that target capture combined withamplification and detection was able to detect as few as one copy of theLegionella target per reaction, although results between duplicatesamples were more variable for samples with lower copy numbers (1-10copies) than for samples that contained 100 or more copies.

TABLE 5 Detection of L. pneumophila 16S rRNA Following Target Captureand Amplification Target Copies Target Capture in Sample SEQ ID NO: 67Target Capture SEQ ID NO: 66 1 971,164 960,828 10 974,831 1,888,180 1001,108,534 1,972,797 10000 1,991,759 1,977,446

Similar experiments were performed as described above in assays thatused the same target capture probe, amplification primers, and detectionprobe, but using target RNA prepared from cultures of L. pneumophilaserotype 1, L. pneumophila serotype 799, L. longbeacheae, E. coli, S.pyrogenes, Enterococcus sp., S. agalactiae and S. aureus, as describedin Example 2. The results showed that the assay detected specifically L.pneumophila of both serotypes 1 and 799 (4.9-5.3×10⁶ RLU detected), butdid not detect positive signals for any of the other target nucleicacids isolated from other bacteria (all less than 10,000 RLU, usually atbackground level of about 2000 RLU).

Amplification and Detection of 23S rRNA Sequences of L. pneumophila

For amplification and detection of sequences found in 23S rRNA sequences(which include 23S rRNA or DNA encoding 23S rRNA) of L. pneumophila,oligomers were designed that act as amplification oligomers anddetection probes by comparing known sequences of 23S rRNA or genesequence encoding 23S rRNA and selecting sequences that are common to L.pneumophila isolates, but preferably are not completely identical to 23SrRNA sequences of other Legionella species or other bacteria. Sequencecomparisons were conducted by using known 23S rRNA sequences (RNA orgenes) of Legionella species (L. anisa, L. briminghamiensis, L.bozemanii, L. cherrii, L. dumoffii, L. gormanii, L. hackeliae, L.israelensis, L. jamestowniensis, L. jordansis, L. longbeachae, L.micdadei, L. oakridgenesis, L. parisiensis, L. pneumophila, L.rubrilucens, L. santicrucis, L. sainthelensi, and L. wadsworthii) and ofother bacteria (Acinetobacter calcoeceticus, Enterobacter aerogenes, E.cloacae, E. gergoviae, Pseudomonas aeruginosa, P. alcaligenes, P.cepacia, P. fluorescens, P. maltophilia, P. mirabolis, P. vulgaris, P.stutzeri, Corynebacterium diversus, C. pseudotuberculosis, Klebsiellapneumoniae, K. rhinoscleromatis, K. oxytoca, Salmonella typhimurium, S.enteritidis, Shigella sonnei, and Vibrio parahaemolyticus). Specificsequences were selected, synthesized in vitro, and the L. pneumophilaoligomers were characterized to determine their Tm and hybridizationcharacteristics with complementary target sequences (synthetic orpurified rRNA from bacteria) by using standard laboratory methods.Selected L. pneumophila oligomer sequences were further tested by usingdifferent combinations of amplification oligomers in amplificationreactions with synthetic 23S RNA target sequences or 23S rRNA purifiedfrom various Legionella species grown in culture to determine theamplification efficiencies for 23S rRNA target sequences. The relativeefficiencies of different amplification oligomer combinations weremeasured by detecting the amplified products of the reactions by bindinga labeled probe to the amplified products and detecting the relativeamount of signal that indicated the amount of amplified product. Usuallyinitial testing of amplification efficiency involved detection of theamplified products by using an AE-labeled linear detection probehybridized to amplified products and detected by using a HPA method thatselectively degrades the AE in unhybridized probes and detects signalfrom hybridized probes (U.S. Pat. Nos. 5,283,174, 5,656,207, 5,658,737and 5,824,475).

Selected amplification oligomers for 23S rRNA target sequences are shownin Table 6, in which lower case letters are used for the promotersequences in promoter primer and promoter provider oligomers. Table 6lists oligomers that consist of target-specific sequences that areidentical to those in corresponding promoter primers (e.g., thetarget-specific sequence of SEQ ID NO:71 is included in the threepromoter primers of SEQ ID Nos. 75-77). Those skilled in the art ofmolecular amplification methods will appreciate that a target-specificsequence may be synthesized with any known promoter sequence attached tothe 5′ end of the target-specific sequence. Preferred embodimentsinclude a promoter specific for T7 RNA polymerase, as shown in SEQ IDNos. 90, 91, and 92.

TABLE 6 Amplification Oligomers for 23S rRNA Target Sequences SEQSequence ID NO. CACGTGTCCCGGCCTACTTGTTCG 69aatttaatacgactcactatagggagaCACGTGTCCCGGCC 70 TACTTGTTCGCTGAGTAGAACAATTTGGGAAAGTTGGCG 71 CUGAGUAGAACAAUUUGGGAAAGUUGGCG 74aatttaatacgactcactatagggagaCTGAGTAGAACAAT 75 TTGGGAAAGTTGGCGatttaatacgactcactatagggagaCTGAGTAGAACAATT 76 TGGGAAAGTTGGCGtttaatacgactcactatagggagaCTGAGTAGAACAATTT 77 GGGAAAGTTGGCGGGGAAAGTTGGCGATAGAGGGTGAAAGCC 78aatttaatacgactcactatagggagaGGGAAAGTTGGCGA 79 TAGAGGGTGAAAGCCGGAGCCTGGCGTGATTTATTATTGAACTGAG 80aatttaatacgactcactatagggagaGGAGCCTGGCGTGA 81 TTTATTATTGAACTGAGatttaatacgactcactatagggagaGGAGCCTGGCGTGAT 82 TTATTATTGAACTGAGtttaatacgactcactatagggagaGGAGCCTGGCGTGATT 83 TATTATTGAACTGAGCUCAGUUCAAUAAUAAAUCACG 84 CUUUCCCAAAUUGUUCUACUCAG 85 GCUCCUCCCCGUUCGCUC86 GGAUTTCACGTGTCCCGGCCTACTTG 87

Probes specific for amplified products of 23S rRNA sequences made byusing combinations of the amplification oligomers shown in Table 6include those of SEQ ID NO:72 (CGAAGGUUUGAUGAGGAAC), SEQ ID NO:88(cCCUCAUCAAACCUUCGUAgaggg), and SEQ ID NO:89 (CGUGCCUAGUUCCUCAUCgcacg),in which lower case letters are used for the torch closing means thathybridize to the torch's target detecting means (i.e. thetarget-complementary region) in the absence of the target sequence. Thetarget-complementary regions of SEQ ID NO:88 and SEQ ID NO:89 areaccordingly SEQ ID NO:97 (CCUCAUCAAACCUUCGUA) and SEQ ID NO:98(CGUGCCUAGUUCCUCAUC), respectively. Preferred embodiments of detectionprobes of SEQ ID NO:72 are labeled with an AE label attached to theoligomer by a non-nucleotide linker at positions between nucleotides 6and 7, 8 and 9, or 12 and 13. Preferred embodiments of the probes of SEQID Nos. 88 and 89 include a 5′ fluorophore (e.g., fluorescein), a 3′quencher (e.g., DABCYL), and an abasic moiety (e.g., C9) betweennucleotides 5 and 6. Preferred embodiments of detection probe oligomersmay also include a mixture of DNA and RNA bases, and 2′ methoxy RNAgroups. SEQ ID Nos. 72, 88 and 89 are all 2′ methoxy modified RNA probeoligomers.

Embodiments of capture probes for use in sample preparation to separateLegionella 23S rRNA target nucleic acids from other sample componentsinclude those that contain a target-specific sequence of SEQ ID NO:73(CCGAGTTCGCCTTTGCATCCTATG) that hybridizes to a 23S rRNA sequence or DNAencoding 23S rRNA. Preferred capture probe embodiments include a 3′ tailsequence covalently attached to the target-specific sequence of SEQ IDNO:73, e.g., a dT₃A₃₀ linked to the 3′ end of SEQ ID NO:73, thatfunctions as a binding partner to bind the hybridization complex made upof the Legionella target nucleic acid and the capture probe to animmobilized probe on a support.

Different amplification oligomers combinations were made from thoselisted in Table 6 and were tested in single primer transcriptionassociated amplifications as described above, using total RNA or 23SrRNA isolated from L. pneumophila and other bacteria as target nucleicacid. Amplified products were detected by using hairpin probes (torch ormolecular beacon probes) labeled with a fluorophore (5′ fluorescein) and3′ quencher (DABCYL), detecting the fluorescence emitted when the probebound to amplified sequences. Those assays specifically amplified anddetected L. pneumophila sequences with a sensitivity of 10⁻¹⁰ M copiesper reaction.

Example 5: Amplification and Detection of L. pneumophila 23S rRNA TargetSequence

Amplification and detection of a L. pneumophila 23S rRNA target sequencewas demonstrated in real time by using a probe that hybridizes to theamplified product during the amplification reaction. Amplification wasperformed by using a single primer transcription associatedamplification procedure substantially as described in detail in US2006-0046265 A1, conducted by using some of the selected amplificationoligomers. Amplification oligomer combinations tested included (1)primer of SEQ ID NO:87 with promoter provider of SEQ ID NO:79 andblocker oligomer of SEQ ID NO:85, or (2) primer of SEQ ID NO:87 withpromoter provider of SEQ ID NO:75 and blocker oligomer of SEQ ID NO:84.Each of the assays was performed in an amplification reaction (0.060 mltotal volume) that contained the L. pneumophila target RNA andamplification reagents substantially as described for TMA reactions butwith a promoter provider oligomer (12 pmol per reaction), a primeroligomer (12 pmol per reaction), and a blocker oligomer (0.8 pmol perreaction) and a hairpin probe (molecular torch) of SEQ ID NO:89.Reaction mixtures containing the amplification oligomers, target andamplification reagents (but not enzymes) were covered to preventevaporation, incubated 5 min at 95° C., then 2 min at 42° C., thenenzymes were added (10 μl vol) and the reactions were mixed andincubated for 30 min at 42° C., measuring fluorescence every 30 secduring the amplification reaction after enzyme addition. Results ofthese tests showed that both combinations of amplification oligomersperformed well and the hairpin probe provided detectable signalgenerally at by the 15^(th) to 25^(th) predetermined interval afteramplification began. The assays detected 100 or more copies of thetarget nucleic acid.

Similar tests were performed by using the L. pneumophila 23S rRNA targetwith a combination of a primer oligomer of SEQ ID NO:87, a promoterprovider oligomer of SEQ ID NO:75, and a blocker oligomer of either SEQID NO:84 or 85. Amplified products were detected by using a hairpinprobe of SEQ ID NO:88. Results of these tests showed that the probe ofSEQ ID NO:88 detected 10³ or more copies of the target nucleic acid.

A preferred combination of amplification oligomers for real timedetection of L. pneumophila 23S rRNA target determined by these testsincluded those of SEQ ID Nos. 75, 84 and 87. Preferred methods ofamplifying and detecting 23S rRNA of L. pneumophila also include atarget capture step performed substantially as described above forcapture of 16S rRNA of L. pneumophila, but using a target captureoligomer specific for 23S rRNA sequences, such as an oligomer thatincludes SEQ ID NO:73. Preferred methods use a capture probe of SEQ IDNO:73 synthesized with 2′ methoxy RNA groups in the target-specificsequence and a covalently linked to 3′ tail sequence, e.g., dT₃A₃₀.Using these assays, L. pneumophila of serogroups 1 to 14 (ATCC accessionnos. 33152, 22154, 22155, 33156, 33215, 33823, 35096, 35289, 43283,43130, 43290, 43736, and 43703) were positively detected when RNA fromabout 6×10⁵ cells per 0.1 ml were tested, but no cross-reactivity wasobserved when similar samples were prepared from 14 Legionella species(non-L. pneumophila species) and 28 non-Legionellae bacteria and tested.The tested non-L. pneumophila species included L. feelei, L.longbeachae, L. wadsworthii, L. dumoffii, L. haeckeliae, L.oakridgeensis, L. birminghamensis, L. jamestownensis, L. jordanis, L.rubrilucens, L. micdadei, L. parisiensis, L. gormandii, and L.bozemanii. The tested non-Legionellae bacteria included Pseudomonasputida, P. cepacia, P. stutzeri, P. acidoverans, P. alcaligenes, P.auroginosa, P. medocina, Acinetobacter calcoaceticus, Staphylococcusepidermidis, Klebsiella pneumoniae, Micrococcus catarrhalis,Enterococcus faecalis, Neisseria meningitidis, N. gonoerrhoeae,Escherichia coli, Moraxella ovis, Haemophilus influenzae, H.parainfluenzae, Streptococcus sanguis, S. mutans, S. pyrogene, S.agalactiae, S. pneumoniae, Corynebacterium aquaticum, C. xerosis, and C.striatum.

The invention claimed is:
 1. A method for the amplification ofLegionella-pneumophila 23S nucleic acid present in a sample, the methodcomprising the steps of: A) contacting the sample with a capture probethat hybridizes to a Legionella-pneumophila 23S target nucleic acid andseparating the target nucleic acid away from other components of thesample, wherein said separating comprises removing the other samplecomponents from a Legionella target:capture probe complex attached to aparticulate solid support; B) contacting the separated target nucleicacid from step A with a set of oligonucleotides, the set ofoligonucleotides comprising: (i) a first amplification oligonucleotide,the base sequence of which consists of SEQ ID NO:87; (ii) a secondamplification oligonucleotide, the base sequence of which consists ofSEQ ID NO:71 joined at its 5′ end to a promoter sequence; and (iii) ablocker oligonucleotide, the base sequence of which is at least 80%identical to SEQ ID NO:84; and C) performing a transcription associatedamplification reaction to generate an amplification product from thetarget nucleic acid using the set of oligonucleotides.
 2. The method ofclaim 1, wherein the base sequence of the second amplificationoligonucleotide consists of SEQ ID NO:75.
 3. The method of claim 1,further comprising the step of: D) detecting the amplification productfrom step C in real time by specifically hybridizing the amplificationproduct with a hairpin detection probe under conditions that allowhybridization, wherein the base sequence of the hairpin detection probeconsists of: (i) a target specific sequence that specifically hybridizesto an amplification product target sequence, wherein the target specificsequence is selected from the group consisting of SEQ ID NO:97, SEQ IDNO:98, and the complement of SEQ ID NO:97 or 98, and (ii) a closingsequence, wherein detection of the amplification product indicates thepresence of Legionella pneumophila in the sample.
 4. A reaction mixturefor performing the isothermal amplification of a Legionella pneumophila23S nucleic acid, wherein the reaction mixture comprises: A) aLegionella pneumophila 23S target nucleic acid that has been separatedfrom components of a biological sample derived from a human; B) a firstamplification oligonucleotide, the base sequence of which consists ofSEQ ID NO:87; C) a second amplification oligonucleotide, the basesequence of which consists of SEQ ID NO:71 joined at its 5′ end to SEQID NO:90; D) a blocker oligonucleotide, the base sequence of which is atleast 80% identical to SEQ ID NO:84; and E) a particulate solid support.5. A method for the detection of Legionella pneumophila 23S nucleic acidpresent in a sample, the method comprising the step of steps of: A)contacting the sample with a capture probe that hybridizes to aLegionella pneumophila 23S target nucleic acid and separating the targetnucleic acid away from other components of the sample; B) contacting theseparated target nucleic acid from step (A) with a set ofoligonucleotides, the set of oligonucleotides comprising: (i) a firstamplification oligonucleotide, the base sequence of which consists ofSEQ ID NO:87, and (ii) a second amplification oligonucleotide, the basesequence of which consists of SEQ ID NO:71 joined at its 5′ end to apromoter sequence; and (iii) a blocker oligonucleotide, the basesequence of which is at least 80% identical to SEQ ID NO:84; C)performing a transcription associated amplification reaction to generatean amplification product from the target nucleic acid using the set ofoligonucleotides; and D) detecting the amplification product from step Cin real time by specifically hybridizing the amplification product witha hairpin detection probe under conditions that allow hybridization,wherein the base sequence of the hairpin detection probe consists of:(i) a target specific sequence that specifically hybridizes to anamplification product target sequence, wherein the target specificsequence is selected from the group consisting of SEQ ID NO:97, SEQ IDNO:98, and the complement of SEQ ID NO:97 or 98, and (ii) a closingsequence, wherein detection of the amplification product indicates thepresence of Legionella pneumophila in the sample.
 6. The method of claim5, wherein the base sequence of the 5′ promoter sequence consists of thebase sequence of SEQ ID NO:90.
 7. The method of claim 5, wherein thetarget specific sequence and the closing sequence are a sequenceselected from the group consisting of SEQ ID NO:88, SEQ ID NO:89, andthe complement of SEQ ID NO:88 or
 89. 8. The method of claim 7, whereinthe hairpin detection probe further comprises at least one labelselected from the group consisting of: a fluorescent label, a quencherlabel, a chemiluminescent label, a fluorescein label, a DABCYL label andcombinations thereof.
 9. The method of claim 7, wherein the hairpindetection probe further comprises a label pair selected from the groupconsisting of: a F/Q pair, a F/DABCYL pair, a fluorescein/Q pair, aluminescent/adduct pair, a Forrester energy transfer pair, andfluorescein/DABCYL pair.
 10. The method of claim 9, wherein one memberof the pair is attached to the 5′ end of the hairpin detection probe andanother member is attached to the 3′ end of the hairpin detection probe.11. The method of claim 5 wherein the hairpin detection probe furthercomprises a label pair selected from the group consisting of: a F/Qpair, a F/DABCYL pair, a fluorescein/Q pair, a luminescent/adduct pair,a Forrester energy transfer pair, and a fluorescein/DABCYL pair.
 12. Themethod of claim 11, wherein one member of the pair is attached to the 5′end of the hairpin detection probe and another member is attached to the3′ end of the hairpin detection probe.
 13. The method of claim 5,wherein the hairpin detection probe base sequence contains one or moresubstituted sugar moiety selected from the group consisting of 2′methoxy substitutions and 2′ halide substitutions.
 14. The method ofclaim 13, wherein the hairpin detection probe further comprises at leastone label selected from the group consisting of: a fluorescent label, aquencher label, a chemiluminescent label, a fluorescein label, a DABCYLlabel and combinations thereof.
 15. The method of claim 13, wherein thehairpin detection probe further comprises a label pair selected from thegroup consisting of: a F/Q pair, a F/DABCYL pair, a fluorescein/Q pair,a luminescent/adduct pair, a Forrester energy transfer pair, and afluorescein/DABCYL pair, and optionally one member of the pair isattached to the 5′ end of the hairpin detection probe and the othermember is attached to the 3′ end of the hairpin detection probe.
 16. Themethod of claim 5, wherein the base sequence of the second amplificationoligonucleotide consists of the base sequence of SEQ ID NO:75; and thebase sequence of the hairpin detection probe consists of the basesequence of SEQ ID NO:88 or the base sequence of SEQ ID NO:89.